Otolith radiocarbon signatures provide distinct migration history of walleye pollock around Hokkaido, Japan in the North‐Western Pacific

Abstract Trace elements and stable isotope ratios in otoliths have been used as proxies for the migration history of teleosts; however, their application in oceanic fishes remains limited. This study reports the first use of radiocarbons in otoliths to evaluate the horizontal migration histories of an oceanic fish species, the walleye pollock Gadus chalcogrammus. We conducted radiocarbon analyses of three stocks sourced from Hokkaido, Japan. The radiocarbon concentrations from the outermost portion of the otoliths from the Japanese Pacific, Northern Japan Sea (JS), and Southern Okhotsk Sea (OS) stocks were in general agreement with the seawater radiocarbon concentration of the sampling region, suggesting that pollock of all three stocks generally inhabited the within the sea region where each pollocks were sampled throughout their life cycle. However, the radiocarbon signals also provided some indications that some JS and OS stocks may be migrating between different sea regions. The proposed novel approach of reconstructing the individual migration history of marine fish using radiocarbon in otoliths may help examine fish migration with a higher temporal and spatial resolution that could not be achieved by trace elements and stable isotope ratios.

Accurate and reliable stock identification data are essential for successful fishery management.Although each population group should be managed separately for an optimized outcome (e.g., Begg et al., 1999), the mixing of stocks owing to the ontogenetic migration of species frequently confounds the stock structure.Understanding the migratory history of each stock is beneficial for natural resource conservation.Previous studies have used trace elements and stable isotope ratios of fish tissues as "natural tags" to trace fish migration (Kubota et al., 2015;Tzadik et al., 2017;West et al., 2006).In particular, metabolically inert calcified structures, the otolith, have been widely studied (Amekawa et al., 2016;Campana, 1999;Sturrock et al., 2012).Their chemical composition predominantly reflects that of ambient water at the time of calcification (Campana et al., 1994).
Unlike other calcified structures such as endoskeletons or fin rays (Campana et al., 2000;Tzadik et al., 2017), otoliths are not resorbed after formation, and can thus be used as a permanent fingerprint of a fish's life history.Otoliths are composed of >95% calcium carbonate (Campana, 1999), with protein matrices making up the remaining mass.The major elements in otoliths are therefore calcium, carbon, and oxygen, with 47 other minor or trace elements incorporated in the form of proteins or calcium carbonate lattices.Despite the diversity of minor and trace elements in the fish otoliths, most elements and stable isotopes have limited potential as migration proxies for oceanic fish.The horizontal gradients of these chemical properties are generally small or negligible in oceanic waters, except in nearshore regions, which are influenced by terrestrial sources (Kubota et al., 2015;Yokouchi et al., 2017).It is analytically challenging to resolve the small chemical signals of migration in the otoliths of oceanic fish (Proctor et al., 1995;Sturrock et al., 2012;Yokouchi et al., 2017).
Furthermore, owing to different physiological processes, most elements show interspecific variability in relationships between otolith compositions and ambient water element concentrations (Brown & Severin, 2009;Rooker et al., 2004).Physiological effects, such as age, somatic growth rate, and gonadal maturation, also affect elemental concentrations in otoliths, which further confounds elemental composition (Kalish, 1991;Sturrock et al., 2014).Therefore, it is necessary to identify a proxy that exhibits high spatial heterogeneity and is unaffected by biological fractionation.
Recently, radiocarbon ( 14 C) has emerged as a potentially useful tracer for examining the migration and feeding history of marine fish and mammals (Eisenmann et al., 2017;Larsen et al., 2018;Miyairi et al., 2023).In otoliths, radiocarbon is used to confirm the age of fish (Andrews et al., 2011;Campana, 1997;Kalish et al., 1996).
As otolith carbon is largely derived from ambient water (Solomon et al., 2006), it is possible to derive the year of birth of fish by comparing the radiocarbon of the otolith core with that of seawater.One advantage of using 14 C as an ecological tracer is that 14 C content is reported as Δ 14 C corrected for isotope fractionation (Stuiver & Polach, 1977).Unlike conventional tracers, Δ 14 C is a proxy that is purely dependent on its source (for the calcium carbonate in otoliths, mainly dissolved inorganic carbon (DIC) in seawater), without the confounding effects of animal physiology and isotope fractionation (Larsen et al., 2018).The large geographic gradient of Δ 14 C values of DIC in the upper oceans constitutes another substantial advantage for 14 C as a tracer (Lan et al., 2023;Servettaz et al., 2019). 14C produced in the upper atmosphere enters the ocean through the air-sea gas exchange of carbon dioxide and is transferred to deep waters by global ocean circulation (McNichol & Aluwihare, 2007).
Because it is subject to radioactive decay (half-life of 5730 years), 14 C is depleted in deep waters that have been isolated from the airsea gas exchange for decades or centuries.In low-to middle-latitude oceans where the thermocline is steep, surface waters are isolated from 14 C depleted subsurface waters and display high Δ 14 C values.
In contrast, in upwelling regions, the intrusion of old water from deep layers results in a decrease in Δ 14 C values in the upper layers (Toggweiler et al., 2019).Nuclear weapons testing in the 1950s and 1960s, which rapidly increased atmospheric and surface ocean Δ 14 C values, magnified the gradient of Δ 14 C in the upper ocean (Kumamoto et al., 2013;McNichol & Aluwihare, 2007).Although this artificially introduced Δ 14 C has been decreasing due to absorption by atmosphere-ocean mixing, significant "bomb-peak" signatures remain in upper oceans in the present time.Currently, in the western North Pacific, the western boundary current (Kuroshio current) that originates from the low-latitude western Pacific is enriched in 14 C (ca. 50‰: Ishikawa et al., 2021;Yokoyama et al., 2022).In contrast, the subarctic current (Oyashio current) delivers water with low Δ 14 C values of ca.−50‰ (Ishikawa et al., 2021;Ota et al., 2021;Satoh et al., 2019).Therefore, a large geographic gradient of Δ 14 C (−50 to 50‰) appears in oceanic regions influenced by these two current systems.These regions include those around Hokkaido, where a large amount of fishing activity takes place.Similarly, geographic Δ 14 C gradients appear in other oceanic regions influenced by upwelling (e.g., off California and the Southern Ocean; Druffel & Williams, 1991;Toggweiler et al., 2019).Although a strong geographic gradient of Δ 14 C can serve as a potentially powerful marker to examine the horizontal migration of marine organisms (Eisenmann et al., 2017), no previous studies, to the best of our knowledge, have explored the use of otolith radiocarbon to investigate the horizontal migration of fish.
The purpose of this study was to explore the potential application of otolith radiocarbon to reconstruct the migration history of marine organisms using walleye pollock (Gadus chalcogrammus), hereafter referred to as pollock, as a model.Pollock is a key species in the North Pacific ecosystem (Bailey et al., 1999) and an important fishery resource (Springer, 1992).It forms meta-populations around Hokkaido, separating in four fishery stocks: the Japanese Pacific (JP), the Northern Japan Sea (JS), the Southern Okhotsk Sea (OS), and the Nemuro Strait stocks (Mori & Hiyama, 2014;Watanobe, 2008).
Previous work has found that the annual stock distribution of pollock changes dramatically depending on environmental factors such as temperature and food availability (Maeda et al., 1993).Early tagging survey results suggest that some exchanges of individuals take place across different regions of Hokkaido (Nishimura et al., 2002;Yoshida, 1982).However, the ontogenetic migration histories of these individuals remain unclear.Understanding the migration patterns of each stock in Hokkaido is essential for supporting appropriate fishery management.

| Sample collection
Pollock were collected using mid-water/bottom trawls (RV Kaiyo-Maru No. 5 and RV Hokko-Maru cruises) from three regions (JP, JS, and OS) around Hokkaido (Figure 1b, Table A1).After measuring the body length (BL; length from the tip of the lower jaw to the base of the caudal fin) and weight (BW) of the specimens, pairs of sagittal otoliths were removed.Otoliths removed from the large (>400 mm BL) size group, corresponding to an approximately 5-year-old or older age class (Hamatsu et al., 2004;Kooka, 2012), were used for radiocarbon analyses (Table 1).Pollock collected from the JP (n = 5), JS (n = 5), and OS (n = 4) regions were named as Pacific-1 to 5, Japan-1 to 5, and Okhotsk-1 to 4, respectively.
Eight to 10 discrete gas samples were collected from each otolith before dissolving the entire otolith.The reaction time for each gas collection time was determined by the otolith weight so that an approximately equal amount of CO 2 gas (3-6 mg C depending on the size of the otolith) was collected at each step.Then the CO 2 gas was cryogenically trapped in a vacuum line, reduced to solid graphite (Yokoyama et al., 2022), and analyzed using a single-stage accelerator mass spectrometer at the Atmosphere and Ocean Research Institute at The University of Tokyo, Japan (Yokoyama et al., 2019).
The carbon obtained using the stepwise acid dissolution method was derived solely from calcium carbonate.As phosphoric acid reacts only with calcium carbonate, the remaining protein is preserved in the phosphoric acid solution.This method is typically used in carbonate radiocarbon analysis including that of otoliths (e.g., Burr et al., 1992;Grammer et al., 2015;Miyairi et al., 2023;Yokoyama et al., 2000).
The stepwise dissolution procedure of otoliths (Miyairi et al., 2023) yields a series of discrete gas collections from the outer portion (1st sample collection) to the inner core (nth sample collection) (Table A2).To evaluate the life stage represented by each gas sample, we calculated the cumulative amount of gas normalized by total gas collected in inverse order of gas sample collection (for the convenience of data presentation) using the following equations: is the Δ 14 C of the kth gas collection, g k is the amount of CO 2 (hPa) obtained in the kth gas collection and n is the total number of CO 2 gas collections from one otolith sample (n = 10 for Japan-1,2,5, Pacific-1,2,3, and Okhotsk-1,3,4; n = 9 for Japan-4, Pacific-4, and Okhotsk-2; n = 8 for Japan-3 and Pacific-5), with an error of x n expressed as ± This would allow the timing of otolith formation to be estimated.As the gas was not collected in equal amounts, this would provide a more plausible estimate of the timing of otolith formation than plotting individual measurement points at equal intervals.The life stage was scored using x values, with the entire lifespan scaled between 0 (innermost core portion) and 100 (outermost edge) %.The plot of Δ 14 C values of discrete gas samples over x values (life stage) for

| RE SULTS
The Δ 14 C values of the outermost portion of otoliths were −50.55 ± 4.93, 24.57 ± 3.78, and −25.64 ± 9.78 for the JP, JS, and OS regions, respectively (Table 2).Because the outermost portion of samples generally contributed approximately 10% of the collected CO 2 gas (except for one individual in the JP stock [Pacific-3] which contained 5.7% of total gas in the outermost portion), the values represent the mean Δ 14 C value of ambient seawater DIC in which each fish had spent the final 10% of their life history.This is roughly equivalent to 0.5 years if the pollock was 5 years old at the time of sample collection.However, the time resolution of the otolith radiocarbon records should be interpreted with caution (see Discussion).
We obtained 8-10 Δ 14 C values for each otolith using a stepwise dissolution method (Table A3), representing a time series across the individual's entire lifetime (the otolith radiocarbon record).The otoliths from the JP stock were strongly depleted in radiocarbon (range, −70‰ to −40‰) throughout their lives (Figure 2), and means differed among the five individuals (Table 3).In contrast, otoliths from the five individuals of the JS stock were enriched with radiocarbon (range, 20‰-40‰), except that some gas samples collected from Japan-1 and Japan-5 displayed anomalously low values (−20‰ to −50‰) at specific life stages (Figure 3).Specifically, in Japan-1, Note: In the sex column, "F" represents female and "M" represents male pollock.Details of trawl locations are shown in Table A1.
Mean ranks of Δ 14 C values of otoliths significantly differed among these individuals as well (Table 4).
The patterns in the otolith radiocarbon records for the four individuals collected from the OS region were more diverse than those from other regions (Figure 4).reported in the corresponding regions or water masses (Aramaki et al., 2001(Aramaki et al., , 2007;;Satoh, 2020;Satoh et al., 2019).Previous work has found that in the Oyashio-influenced region (the JP region), the median Δ 14 C value (interquartile range) was −42‰ (−34 to −60) [estimated from the data reported by Satoh et al. (2019) and Satoh (2020) for the depth of 0-200 m], close to the range of Δ 14 C values for the otolith outermost portion of the JP stock (c.2).Aramaki et al. (2007) investigated the Δ 14 C values of seawater in the Sea of Japan (the JS region), reporting that seawater Δ 14 C ranged from 20% to 70‰ in the upper layer.Although the upper range of these measurements exceeded the range we observed in the outermost otolith portion of the JS stock (Figure 3), this could be due to the timing of sample collection.As Aramaki et al.'s samples were collected in 1998, Δ 14 C values were higher than at present due to the more substantial effects of bomb carbon in water masses originating from the subtropical gyre (Kumamoto et al., 2013).In the Okhotsk Sea (the OS region), a study conducted in the Kuril Islands found that seawater Δ 14 C values mostly ranged from −60% to 0‰ in the upper layer (0-200 m) (Aramaki et al., 2001).These values are intermediate to those reported for the Oyashio current (Satoh, 2020;Satoh et al., 2019) and the Sea of Japan (Aramaki et al., 2007), roughly corresponding to the range of otolith outermost portion Δ 14 C values of the OS stock (Figure 4).Although a rigorous comparison would require the simultaneous sampling of pollock and seawater, the available data suggest that the radiocarbon content of the outermost otolith portion agreed reasonably well with ambient seawater radiocarbon signatures.

| Time resolution of each step and its associated uncertainties
Using a stepwise dissolution approach, we successfully obtained time-series records of otolith radiocarbon.Assuming that each gas sample corresponded to an equal length of otolith lifespan, the time resolution of our chronological reconstruction was 0.5-0.6 years (i.e., the age of fish (5 years) divided by eight, nine, or 10).Note that the time resolution range was an approximate value.Otolith growth may decrease with fish age due to declining somatic growth (Hanson & Stafford, 2017), implying that gases collected from the outer portion of the otolith have a lower time resolution (that is, integrate information over a longer period) than those collected from the inner core.The complex morphology of otoliths adds another uncertainty Otolith radiocarbon records of walleye pollock from Sea of Japan region (n = 5).Iconography is as in Figure 2. One measurement point from Japan-1, from Japan-3, and three from Japan-5 were excluded due to sample loss or failure in AMS measurement.

TA B L E 4
Mean of the outermost otolith Δ 14 C for Sea of Japan walleye pollock specimens analyzed in this study.
to the estimation of the time resolution, as the growth rate of otoliths can differ depending on their axes (Galley et al., 2006;Gauldie & Nelson, 1990).Future studies should examine the relationship between radiocarbon signatures and pollock growth estimated from otolith readings with the aid of the otolith micro-milling technique (Høie et al., 2004).

| Resolving fish migration history: Did pollock move to different regions?
Five individuals from the JP stock displayed stable radiocarbon values, indicating that they remained in the JP region throughout their lives.Our findings are consistent with the results of previous studies suggesting that pollock have several spawning grounds in the JP region, displaying dispersed feeding and homing migration within the region (Figure 1, Nishimura et al., 2002).Interestingly, mean ranks of Δ 14 C values differed significantly among JP stock individuals (Table 3), likely reflecting differences in the location of spawning ground, migration trajectory, or both, within the JP region.
Future studies examining the fine-scale distribution patterns of seawater radiocarbon in the JP region would be helpful in resolving the differentiation in migratory behavior within the region among JP individuals.
The otolith radiocarbon data for the JS stock individuals provided distinct signatures, suggesting that the two individuals from this stock migrated across regions (Figure 3).While three individuals (Japan-2,3,4) with stably high Δ 14 C values likely remained in the JS region throughout their lives, anomalously low Δ 14 C values recorded for Japan-1 and Japan-5 indicate that they were exposed to radiocarbon-depleted seawater at certain stages of their lives (Figure 3).For Japan-1, the decrease of Δ 14 C from 34.62‰ to −22.77‰ at x = 66.32%, followed by a return to the original level of 27.11‰, indicates that this individual moved temporarily from JS to probably the OS region during its adult stage.Similarly, Japan-5 appears to have stayed in a Δ 14 C-depleted region (likely JP) when it was young (x = 12.19%), migrated to the JS region after growing to maturity (x = 44.25%),re-visited JP at x = 57.69%,and then remained in JS for the rest of its life.These inferences on migration destinations are based on the similarity of Δ 14 C values between each otolith section and the regional radiocarbon signature.That is, anomalous Δ 14 C values of −22.77‰ and ca.−40‰ to −50‰ for Japan-1 and Japan-5 respectively correspond to the intermediate (OS) and low (JP) Δ 14 C signatures of seawater, respectively (Table 2).However, this destination assignment is subject to uncertainty, especially for Japan-1, because the radiocarbon signal recorded in the destination region was "diluted" by the Δ 14 C signature in the home region if the staying period in the destination region was less than the time resolution of our record (approximately 6 months).This means that the intermediate Δ 14 C signal in Japan-1 can be explained by either a short visit to the JP region (low Δ 14 C) or a long visit to the OS region (intermediate Δ 14 C).This should be solved in future studies by improving the temporal resolution of otolith radiocarbon records.

F I G U R E 4
Otolith radiocarbon records of walleye pollock from Southern Okhotsk Sea region (n = 4).Iconography is as in Figure 2. Two measurement points from Okhotsk-3 were excluded due to failure in AMS measurement.

TA B L E 5
Mean of the outermost otolith Δ 14 C for Okhotsk Sea walleye pollock specimens analyzed in this study.Diverse patterns were found in the otolith radiocarbon records of individuals collected from the OS region, for which two possible explanations exist that are not mutually exclusive.First, the OS stock may consist of individuals with diverse migratory behaviors, including those who tend to stay in the OS region (Okhotsk-1), those who display temporary migration to the JS region with a high radiocarbon signature (Okhotsk-3), and those who tend to migrate between the OS and JP regions (Okhotsk-4).Second, the distribution of seawater radiocarbons in the OS region might be more variable across the season, across localities, or both, than in other regions.For example, the seasonal exchange of seawater between adjacent regions and local upwelling could introduce large spatial and temporal heterogeneities in radiocarbon distributions even at a scale of a few to tens of kilometers, depending on the oceanographic settings (Satoh, 2020).
Such heterogeneities in seawater radiocarbon distribution within a region complicate the interpretation of otolith radiocarbon records.
Improving our understanding of the temporal and spatial dynamics of radiocarbons in target regions will be a major challenge for future studies.
Our interpretation of otolith radiocarbon records as evidence of the migration of Japan-1 and Japan-5 across regions (horizontal migration) may be challenged by the proposition that the otolith radiocarbon record reflects the vertical movement of pollock within the same region given that the radiocarbon content of seawater generally decreases with depth (Larsen et al., 2018;Miyairi et al., 2023).We argue against this proposition based on the known vertical migration pattern of pollock and Δ 14 C pattern observed in the individuals analyzed in our study.Pollock ontogenetically change their distribution depth (Honkalehto et al., 2010).
Around Hokkaido, the mean habitat depths (average of night and daytime habitat depth) for juvenile and adult pollock are approximately 50-100 m and 200 m, respectively (Honda et al., 2004;Itaya et al., 2009;Kooka et al., 1998;Miyashita et al., 2004;Shida, 2001).This estimated habitat depth difference between juveniles and adults is equivalent to the depth-dependent offset of 20%-30‰ Δ 14 C in seawater (Aramaki et al., 2007).Therefore, the ontogenetic shift in habitat depth alone would not account for the large shift (60%-80‰) in otolith Δ 14 C observed in Japan-1 and -5.Furthermore, ontogenetic vertical shifts in fish habitat depth generally occur gradually throughout their life (Honda et al., 2004;Itaya et al., 2009), while the migration pattern inferred from the pollock otolith radiocarbon records was rather abrupt.In summary, a large, abrupt change in otolith Δ 14 C cannot be explained by a known ontogenetic vertical migration pattern of pollock.

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Our results corroborate the emerging notion that radiocarbon is a powerful tracer for resolving the migratory behavior of fish (Larsen et al., 2018;Miyairi et al., 2023).The application of the otolith radiocarbon recording approach to pollock around Hokkaido provided new insights into the migration of this enigmatic species across these regions.Most strikingly, the otoliths of some individuals had a record of strong radiocarbon signals that differed from those in their sampling regions, indicating temporary migration to adjacent regions.This finding not only supports previous suggestions based on tagging surveys that there are exchanges of individuals across regional stocks around Hokkaido (Yoshida, 1982) but also provides novel information regarding the migration history of each individual.The number of individuals analyzed for each region (n = 4 or 5) in this study was insufficient to assess the proportion of migrant and non-migrant individuals within each regional stock.Nonetheless, the migration signal detected in two of five (JS stock) and one of four (OS stock) individuals suggests that migration is not rare.Future efforts should focus on increasing the number of individuals analyzed for each stock.In this regard, our stepwise dissolution procedure has an advantage over the micromilling approach in terms of simplicity, cost, and labor intensity (Miyairi et al., 2023).In future studies, there is room for improving the accuracy of time resolution by coherently examining fish age (from otolith readings) and radiocarbon.Furthermore, the use of advanced AMS with an analytical detection limit of 10 μg C per sample promises improvements in the time resolution of the otolith radiocarbon record (Yokoyama et al., 2022).Finally, improving the understanding of radiocarbon distribution patterns at regional and sub-regional scales with the Note: The gas collection rate was calculated from the total gas collection divided by the theoretical amount of gas obtained when assuming that CaCO 3 constitutes 100% of the otolith."1st" to "10th" refers to the amount of gas collected in the first to tenth gas collection (hPa; measurement tube volume, 11.5 mL; therefore, 160 hPa = 1 mg C).N/A: gas collection was not conducted due to the formation of cracks in the otolith.Graphitized gases that did not produce sufficient amount of beam during the measurement (9th collection in Pacific-4 and Japan-5) were excluded in Table A3. |

1
Maps of oceanic region around the study site.(a) General map showing cold, subarctic currents (blue arrows) and warm currents originating from the subtropical region (red arrows) around Japan.Black circles indicate pollock sampling sites.Yellow inverted triangles, pink triangles, and blue squares represent the seawater sampling sites of Aramaki et al. (2007), Aramaki et al. (2001) and Satoh et al. (2019), Satoh (2020), respectively.(b) Close up map of the study area.Black circles indicate the pollock sampling sites.Yellow, blue, and pink shades denote the major distribution areas of JS, JP, and OS stocks, respectively.
each individual was used to infer the time-course changes of otolith Δ 14 C values during its life (see Section 4 for notes on the uncertainty associated with the time resolution of otolith radiocarbon records).Mean ranks of otolith Δ 14 C values among individuals belonging to each stock were compared by the Kruskal-Wallis test followed by the Steel-Dwass test using R v.4.3.1 (R Core Team, 2023).

1
Sampling information of walleye pollock analyzed in this study.

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et al.TA B L E A 3 δ 13 C and Δ 14 C values of walleye pollock otolith analyzed in this study.